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Adipose tissue-derived stem cells (ASCs) hold significant potential for treating various clinical conditions. To enhance their regenerative properties, ASCs can be chemically stimulated using various in vitro protocols. However, unsatisfactory results persist, partly due to the relatively costly long-term methods. Furthermore, current culturing techniques often rely on the use of xenogenic fetal bovine serum that can be immunogenic, limiting clinical translations. To facilitate clinical translation of ASCs-derived therapeutics, the effect of different stimulation protocols on human ASCs cultured in a xeno-free medium (PRIME-XV MSC Expansion XSFM) is investigated. The xeno-free medium was supplemented with stimulants (forskolin (FSK), basic fibroblast growth factor, platelet-derived growth factor-AA, neuregulin-1) in combinations or individually. Stimulation for 72 h in FSK alone, or together with the growth factors, enhanced the production of urokinase plasminogen activator (uPA), a serine protease involved in tissue remodeling processes. Conditioned medium derived from stimulated ASCs enhanced in vitro angiogenesis and endothelial cells migration. This study shows that pro-angiogenic responses in human ASCs can be enhanced with a defined short stimulation protocol using a xeno-free medium. The protocol, using readily available manufacturing cell therapy grade molecules, may boost the regenerative properties of ASCs secretome which could enhance their efficacy in clinical treatments.

Exercise has been shown to promote wound healing, including tendon repair. Myokines released from the exercised muscles are believed to play a significant role in this process. In our previous study, we used an in vitro coculture and loading model to demonstrate that 2% static loading of myoblasts increased the migration and proliferation of cocultured tenocytes─two crucial aspects of wound healing. IGF-1, released from myoblasts in response to 2% static loading, was identified as a contributor to the increased proliferation. However, the factors responsible for the enhanced migration remained unknown. In the current study, we subjected myoblasts in single culture conditions to 2, 5, and 10% static loading and performed proteomic analysis of the cell supernatants. Gene Ontology (GO) analysis revealed that 2% static loading induced the secretion of NBL1, C5, and EFEMP1, which is associated with cell migration and motility. Further investigation by adding exogenous recombinant proteins to human tenocytes showed that NBL1 increased tenocyte migration but not proliferation. This effect was not observed with treatments using C5 and EFEMP1.

Quercetin, a flavonoid known for its antioxidant properties, has recently garnered attention as a potential neuroprotective agent for treatment of the injured nervous system. The repair of peripheral nerve injuries hinges on the proliferation and migration of Schwann cells, which play a crucial role in supporting axonal growth and myelination. In this study we synthesized Quercetin-derived carbon dots (QCDs) and investigated their effects on cultured Schwann cells and the NG108-15 cell line. QCDs was obtained by solvothermal synthesis and characterized via UV–vis absorption spectroscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, and X-ray diffraction analysis. The particles demonstrated significant dose-dependent free radical scavenging activity in DPPH and ABTS radical scavenging assays, supported in vitro proliferation and migration of Schwann cells, expression of neurotrophic and angiogenic growth factors, and stimulated neurite outgrowth from NG108-15 cells. Thus, QCDs could serve as a potential novel treatment strategy to promote regeneration in the injured peripheral nervous system.

Introduction: Before performing cell therapy clinical trials, it is important to understand how cells are influenced by different growth conditions and to find optimal xeno-free medium formulations. In this study we have investigated the properties of adipose tissue-derived stem cells (ASCs) cultured under xeno-free conditions.

Methods: Human lipoaspirate samples were digested to yield the stromal vascular fraction cells which were then seeded in i) Minimum Essential Medium-α (MEM-α) supplemented with 10 % (v/v) fetal bovine serum (FBS), ii) MEM-α supplemented with 2 % (v/v) human platelet lysate (PLT) or iii) PRIME-XV MSC expansion XSFM xeno-free, serum free medium (XV). Flow cytometry for ASCs markers CD73, CD90 and CD105 together with the putative pericyte marker CD146 was performed. Growth rates were monitored over multiple passages and adipogenic differentiation performed at early and expanded passage culture. Growth factor gene expression was analyzed and an in vitro angiogenesis assay performed.

Results: Cells in FBS and PLT grew at similar rates whereas the cells cultured in XV medium proliferated significantly faster up to 60 days in culture. All cultures were >98 % positive for CD73, CD90 and CD105, whereas CD146 expression was significantly higher in XV cells. Adipogenic differentiation was most pronounced in cells which had been cultured in XV medium whilst cells grown in PLT were inferior compared with cells from the FBS cultures. IGF1 gene expression was highest in cells cultured in PLT whilst cells grown in XV medium showed 10-fold lower expression compared with FBS cells. In contrast, HGF gene expression was 90-fold greater in cells cultured in XV medium compared with those cultured in FBS. Conditioned medium from XV cultured cells showed the most angiogenic activity, inducing the greatest endothelial cell network formation and maturation.

Conclusion: Culture under different conditions alters the ASCs characteristics. Since cells cultured in XV medium showed the best adipogenic and angiogenic profile this might be a preferred medium formulation for preparing cells required for reconstructive surgical applications such as cell-assisted fat grafting.

Background: Breast cancer is the most common cancer in women. Cancer cells can persist in a prolonged dormant state for years without any clinical evidence of disease creating an urgent need to better understand the molecular mechanisms leading to relapse. This study aimed to identify extracellular matrix (ECM) components associated with hypoxia-induced breast cancer dormancy. The effects of selected ECM proteins on breast cancer cell proliferation were analyzed, along with their correlation with established prognostic markers in human breast cancer tissue.

Materials and methods: Screening of extracellular matrix proteins was performed in hypoxia-induced dormant MCF-7 breast cancer cells. Proliferation of MCF-7 cells in vitro was subsequently determined in the presence of recombinant ColVII. Adipose tissue-derived mesenchymal stem cells (AdMSCs) subpopulation overexpressing ColVII were indirectly isolated by ColVII receptor integrin-α6 specific antibodies. AdMSCs- MCF-7 3D spheroid cultures were generated to model solid tumour conditions. In addition, the association between ColVII and various prognostic markers was evaluated in clinical samples of human breast cancer tissue.

Results: Dormant MCF-7 cells showed an elevated expression of ColVII while MCF-7 cells cultured on ColVII exhibited reduced proliferation in vitro. In AdMSCs-MCF-7 3D spheroids, a reduced proliferation of MCF-7 cells was observed in Int-α6+/ ColVIIhigh compared with Int-α6-/ ColVIIlow AdMSCs spheroids. In human tissue, high ColVII expression correlated to several positive prognostic markers. Staining for Cytokeratin-5 revealed that ColVIIhigh-expressing cells were predominantly myoepithelial cells.

Conclusion: ColVII is associated with reduced proliferation of breast cancer cells in vitro. ColVII is strongly expressed in myoepithelial cells and in breast cancer tissue the high ColVII expression correlates with several well-known positive prognostic markers, highlighting its potential as a prognostic marker in breast cancer.

Background: Although autologous fat grafting is considered a successful method for the management of contour deformities, the fat graft could potentially induce cancer reappearance by fueling dormant breast cancer cells. Our aim was to characterize the role of adipose-derived stem cells on active and dormant breast cancer cell growth.

Methods: Cobalt chloride was used to induce dormancy in MCF-7 cancer cells. Proliferation of active and dormant cancer cells was determined in the presence of adipose-derived stem cells. A proteome array was used to detect cancer-related protein expression in the cell-conditioned medium. The migration of cancer cells was measured in response to conditioned medium from the adipose-derived stem cells.

Results: The adipose-derived stem cells showed variable effects on active MCF-7 cells growth and inhibited MCF-7 proliferation after the withdrawal of cobalt chloride. Of the 84 different proteins measured in the conditioned medium, only tenascin-C was differentially expressed in the co-cultures. MCF-7 cells alone did not express tenascin-C, whereas co-cultures between MCF-7 and adipose-derived stem cells expressed more tenascin-C versus adipose-derived stem cells alone. The conditioned medium from co-cultures significantly increased the migration of the cancer cells.

Conclusions: Adipose-derived stem cells themselves neither increased the growth or migration of cancer cells, suggesting that autologous fat grafting may be oncologically safe if reconstruction is postponed until there is no evidence of active disease. However, interactions between adipose-derived stem cells and MCF-7 cancer cells could potentially lead to the production of factors, which further promote cancer cell migration.

Background: The use of mesenchymal stem cells (MSCs) for the development of tissue-engineered constructs has advanced in recent years. However, future clinically approved products require following good manufacturing practice (GMP) guidelines. This includes using alternatives to xenogeneic-derived cell culture supplements to avoid rejection of the transplants. Consequently, human platelet lysate (PLT) has been adopted as an affordable and effective alternative to foetal bovine serum (FBS) in traditional 2D cultures. However, little is known about its effect in more advanced 3D culture systems.

Methods: We evaluated bone marrow MSCs (BMSCs) proliferation and CD marker expression in cells expanded in FBS or PLT-supplemented media. Differentiation capacity of the BMSCs expanded in the presence of the different supplements was evaluated in 3D type I collagen hydrogels. Furthermore, the effects of the supplements on the process of differentiation were analyzed by using qPCR and histological staining.

Results: Cell proliferation was greater in PLT-supplemented media versus FBS. BMSCs expanded in PLT showed similar osteogenic differentiation capacity in 3D compared with FBS expanded cells. In contrast, when cells were 3D differentiated in PLT they showed lower osteogenesis versus the traditional FBS protocol. This was also the case for adipogenic differentiation, in which FBS supplementation was superior to PLT.

Conclusions: PLT is a superior alternative to FBS for the expansion of MSCs without compromising their subsequent differentiation capacity in 3D. However, differentiation in PLT is impaired. Thus, PLT can be used to reduce the time required to expand the necessary cell numbers for development of 3D tissue engineered MSC constructs.

Exercise is widely recognized as beneficial for tendon healing. Recently, it has been described that muscle-derived molecules secreted in response to static exercise influence tendon healing. In this study, the optimal static loading intensity for tendon healing and the composition of secretome released by myoblasts in response to different intensities of static strain were investigated. In an in vitro coculture model, myoblasts were mechanically loaded using a Flexcell Tension System. Tenocytes were seeded on transwell inserts that allowed communication between the tenocytes and myoblasts without direct contact. Proliferation and migration assays, together with RNA sequencing, were used to determine potential cellular signaling pathways. The secretome from myoblasts exposed to 2% static loading increased the proliferation and migration of the cocultured tenocytes. RNA-seq analysis revealed that this loading condition upregulated the expression of numerous genes encoding secretory proteins, including insulin-like growth factor-1 (IGF-1). Confirmation of IGF-1 expression and secretion was carried out using qPCR and enzyme-linked immunosorbt assay (ELISA), revealing a statistically significant upregulation in response to 2% static loading in comparison to both control conditions and higher loading intensities of 5% and 10%. Addition of an inhibitor of the IGF-1 receptor (PQ401) to the tenocytes significantly reduced myoblast secretome-induced tenocyte proliferation. In conclusion, IGF-1 may be an important molecule in the statically loaded myoblast secretome, which is responsible for influencing tenocytes during exercise-induced healing.

This is a protocol for a Cochrane Review (Intervention). The objectives are as follows: To assess and compare the effects and complication rates of licensed bioengineered nerve conduits or wraps for surgical repair of traumatic peripheral nerve injuries of the upper limb. To compare effects and complications against the current gold surgical standard (nerve autograft).

Injuries to large peripheral nerves are often associated with tissue defects and require reconstruction using autologous nerve grafts, which have limited availability and result in donor site morbidity. Peripheral nerve-derived hydrogels could potentially supplement or even replace these grafts. In this study, three decellularization protocols based on the ionic detergents sodium dodecyl sulfate (P1) and sodium deoxycholate (P2), or the organic solvent tri-n-butyl phosphate (P3), were used to prepare hydrogels. All protocols resulted in significantly decreased amounts of genomic DNA, but the P2 hydrogel showed the best preservation of extracellular matrix proteins, cytokines, and chemokines, and reduced levels of sulfated glycosaminoglycans. In vitro P1 and P2 hydrogels supported Schwann cell viability, secretion of VEGF, and neurite outgrowth. Surgical repair of a 10 mm-long rat sciatic nerve gap was performed by implantation of tubular polycaprolactone conduits filled with hydrogels followed by analyses using diffusion tensor imaging and immunostaining for neuronal and glial markers. The results demonstrated that the P2 hydrogel considerably increased the number of axons and the distance of regeneration into the distal nerve stump. In summary, the method used to decellularize nerve tissue affects the efficacy of the resulting hydrogels to support regeneration after nerve injury.